There are many procedures performed in life sciences that involve the manipulation of small bioparticles such as cells, bacteria, viruses, protozoa, sperm, eggs, embryos and larvae. Generally the manipulation of these small bioparticles is inherently difficult because the bioparticles are too small to be visualised with the naked eye.
Where one is performing an experiment or procedure that involves adding bioparticles to a vessel (for example, a test tube) there is currently no simple technology available which allows one to know exactly, or at least with a minimal degree of error, how many bioparticles have been added. Typically one would prepare a suspension of the bioparticles and then perform an analysis (for example, enumeration by microscopy or culture on an agar plate), to estimate the number of bioparticles per volume of liquid. An aliquot of this suspension, containing an estimated number of bioparticles, would then be used for a desired purpose; the exact number of bioparticles in the aliquot not being known.
In addition to problems associated with estimation of the numbers of bioparticles by sampling, further problems may result during manipulation of the bioparticles in a particular procedure. For example, an unknown amount of the bioparticles are inevitably lost due to factors such as adhesion to surfaces of vessels or pipettes used, or to denaturation or death of some of the bioparticles. Further, bioparticles can lose their viability or contents over time and accordingly products containing such materials may suffer from a short shelf life. Combined with the above problems, these factors may create gross inaccuracies in experimental data.
A number of products are known which attempt to provide a standardised product having a defined number of bioparticles. However, these products unfortunately fail to address all of the problems above mentioned, and accordingly, may be considered to fall short of providing a desirable product. For example, the degree of error in respect of the number of bioparticles present from one sample of a product to another sample of the same product is likely to be in the order of greater than 50%, in many cases the number of bioparticles present may vary 10 to 100 fold or more.
One example of such known products is Cultiloops® (Oxoid, Australia). Cultiloops® are disposable bacteriological culture loops that contain a loopful of freeze-dried culture of a specific microorganism and are generally used for quality control purposes in microbiology laboratories. While Cultiloops® save time in the preparation of cultures for quality control they unfortunately do not contain accurately defined numbers of cells per loopful. Further, it is possible that a number of the cells present may not be in a viable state.
Several companies supply vials containing an approximate number of microorganisms in a freeze-dried form. Typically, these are manufactured to an accuracy of 1 order of magnitude; for example, a vial will contain between 1000 and 10000 bacteria. To use these products, one generally adds water to the vial to resuspend the freeze-dried microorganisms, subsequently using a pipette to transfer the microorganisms to a sample. Due to the nature of this product, and the means by which it is used, it may not be considered to adequately address the issue of providing accurate and consistent numbers of bacteria, or the issue of the loss of unknown quantities of bacteria during manipulation as a result of adhesion to the side of the vial or the pipette.
BTF Pty Ltd (Australia) markets a product known as EasySeed™ C&G that provides an accurately defined number of inactivated Cryptosporidium and Giardia in liquid in a test tube. While it may be considered that this product overcomes many of the issues associated with providing accurate numbers of microorganisms, during use of the product, an unknown number of the Cryptosporidium and Giardia are generally lost due to adhesion to the side of the test tube or pipette. Further, the cells are not provided in a viable state.
A further example of a presently available product are lenticles, freeze-dried quality control samples prepared by the UK Public Health Laboratory Service (PHLS). Lenticles are prepared by pipetting drops of a viscous bacterial culture a surface and drying the drops to form a lens-shaped freeze-dried pellet. While the lenticules may be considered to overcome the inaccuracies associated with handling liquid quality control samples, they unfortunately do not contain accurately defined numbers of bacteria.
A further product, known as TrueCount® (Becton Dickinson, San Jose, USA), is used in conjunction with flow cytometry to allow one to determine the number of specific cells per millilitre of blood, for example. The product consists of dried balls of approximately 1 mm diameter that contain approximately 50,000 fluorescent beads of approximately 5 μm diameter. While this product may overcome problems associated with the loss of materials during manipulation of a liquid sample, it does not contain an accurately defined number of beads within the dried ball. Further, as the beads do not represent biological-derived material, the product, and the procedure of producing the product, is not concerned with, and therefor may not adequately address, the issue of accuracy or maintenance of viability of the materials.
U.S. Pat. No. 3,932,943 describes a process for the production of a homogeneous, lyophilised product containing at least one biologically active component. The process involves spraying a solution or colloidal suspension containing the biologically active component into a moving bath of fluorocarbon refrigerant, and subsequently lyophilising the resultant frozen droplets. The inventors report that the product has a spherical shape, free-flowing properties, and rapid dissolution times. However, the process does not address the issue of preparing a product that contains accurately defined numbers of bioparticles. In addition, it may be considered that this process does not adequately address the issue of maintenance of bioparticle viability, especially where such bioparticle is a cell.
U.S. Pat. No. 6,106,836 describes a process for the production of a vaccine product comprising a container with freeze-dried vaccine components therein. The process involves the formation of balls containing biological components of estimated numbers utilising the steps of freezing droplets of a suspension containing the biological components in a cryogenic liquid and subjecting them to freeze-drying. The process of this patent does not immediately address the issue of preparing a freeze-dried product that contains accurate numbers of bioparticles. By contrast, products containing estimated numbers of components are made via the above mentioned process, their titre measured, and then a number of products combined, or used to supplement another product, to obtain a desired quantity of components. In addition, the process of U.S. Pat. No. 6,106,836 may not be considered to adequately address the issue of maintenance of the viability of bioparticles during processing. Rather, the process centres on the loss of viability of the bioparticles followed by supplementation of the resultant product with additional viable materials.
Further, U.S. Pat. No. 3,655,838 describes a method for the preparation of pelletised reagents purportedly in a stable, accurate form. In this method, a suspension containing predetermined concentrations of desired reagents is formed into calibrated droplets which are allowed to fall into a liquid having certain characteristics, one of which is a temperature gradient suitable to freeze the droplets. Subsequently, the droplets are dried to form the pelletised product. While the method aims to provide products containing predetermined and pre-tested measured amounts of certain reagents, it may be considered to suffer from inaccuracies in the actual concentration or number of specific components present, due to methods employed to arrive at initial concentration values. Further, the method may be considered not to accurately address the issue of maintenance of viability where the reagent to be processed is a bioparticle.
Current methods for preparing DNA and protein standards for example, typically rely on measuring the absorbance of a solution of DNA or protein and calculating the concentration and then diluting the solution to the desired concentration. These methods do not provide accurate standards.
The present inventors have now developed methods which are capable of producing a desired quanta of bioparticles in products suitable for use as accurate standards for a variety of biological and analytical applications.